Abstract
Technologies to detect the entire bacterial diversity spectra and foodborne pathogens in food represent a fundamental advantage in the control of foodborne illness. Here, we applied high-throughput 16S rRNA sequencing of amplicons obtained by PCR and RT-PCR from extracted DNA and RNA targeting the entire bacterial community and the active bacterial fraction present in some of the most consumed and distributed ready-to-eat (RTE) salad brands in Europe. Customer demands for RTE food are increasing worldwide along with the number of associated foodborne illness and outbreaks. The total aerobic bacterial count in the analyzed samples was in the range of 2–4 × 106 CFU/g (SD ± 1.54 × 106). Culture validated methods did not detect Salmonella spp., Escherichia coli, and other fecal coliforms. 16S rRNA gene Illumina next-generation sequencing (NGS) data were congruent with these culture-based results and confirmed that these and other well-known foodborne bacterial pathogens, such as Listeria, were not detected. However, the fine-resolution of the NGS method unveiled the presence of the opportunistic pathogens Aeromonas hydrophyla and Rahnella aquatilis (relative frequency of 1.33–7.33%) that were metabolically active in addition to non-pathogenic, active members of Yersinia spp. (relative frequency of 0.0015–0.003%). The common ail and foxA marker genes of Yersinia enterocolitica were not detected by qPCR. Finally, our NGS data identified to non-pathogenic Pseudomonas spp. as the most abundant and metabolically active bacteria in the analyzed RTE salads (53–75% of bacterial abundance). Our data demonstrate the power of sequencing, in parallel, both 16S rRNA and rDNA to identify and discriminate those potentially and metabolically active bacteria and pathogens to provide a more complete view that facilitates the control of foodborne diseases, although further work should be conducted to determine the sensitivity of this method for targeting bacteria
Highlights
Ready-to-eat (RTE) raw leafy vegetable-based salads are becoming very popular and widely accepted in our daily diet, and in recent years, these products have clearly covered and satisfied a general growing demand of consumers to incorporate “healthy and green” food
Official outbreaks linked to RTE salads have not been reported in Spain, in other countries, such as USA, two recent multistate outbreaks of E. coli O157:H7 infections linked to fresh lettuce were confirmed, with 272 people infected, 120 hospitalized, and 5 dead since June 2018
total aerobic viable microorganisms (TVC) in all analyzed samples were in the range of 2–4 × 106 bacteria/mL and were quite homogeneous between different samples and brands
Summary
Ready-to-eat (RTE) raw leafy vegetable-based salads are becoming very popular and widely accepted in our daily diet, and in recent years, these products have clearly covered and satisfied a general growing demand of consumers to incorporate “healthy and green” food. Official outbreaks linked to RTE salads have not been reported in Spain, in other countries, such as USA, two recent multistate outbreaks of E. coli O157:H7 infections linked to fresh lettuce were confirmed, with 272 people infected, 120 hospitalized, and 5 dead since June 2018 (official reports from Centres for Disease Control, EEUU). This is only one example, and many more have been reported in other countries [3]. In UK, a national Salmonellosis outbreak was reported [4], and in Switzerland, foodborne transmission of L. monocytogenes was confirmed during 2013–2014 [5]
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